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Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors Min Chen,1,{,{ Kyungah Maeng,1,{ Akbar Nawab,1 Rony A. Francois,1 Julie K. Bray,1 Mary K. Reinhard,2 Sanford L. Boye,3 William W. Hauswirth,3 Frederic J. Kaye,4 Georgiy Aslanidi,5 Arun Srivastava,5 and Maria Zajac-Kaye1,* Departments of 1Anatomy and Cell Biology, 2Veterinary Medicine, 3Ophthalmology, and 4Medicine and 5Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida. { These authors contributed equally to this work. { Current address: Changjiang Scholar Laboratory, Shantou University Medical College, Shantou, Guangdong, China.

Despite efforts to use adeno-associated viral (AAV) vector–mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to KrasG12D+/-, KrasG12D+/-/Pten+/-, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45–70%) compared with WT-AAV8 (7%; p < 0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared with WT-AAV8 (10,285 vs. 1,500 vector copies/lg DNA respectively, p < 0.05). In addition, we observed that intraperitoneal injection of the doublemutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding *14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/lg DNA respectively, p < 0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular. Keywords: gene therapy, pancreatic cancer, adeno-associated virus, AAV8

INTRODUCTION PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) shows a rapid clinical course with a median survival of 6 months and a 5-year survival rate of only 3%.1,2 PDAC responds poorly to conventional therapies, including chemotherapy and irradiation. Surgery is possible only in 10–20% of the patients due to extensive invasion of surrounding structures at the time of diagnosis.3,4 The incidence of pancreatic cancer has increased over the last four decades,5

indicating a pressing need for effective strategies against pancreatic cancer.6,7 Several studies reported the development of recombinant adenoassociated virus (AAV)–mediated gene therapy for PDAC.8,9 For example, AAV2-mediated expression of endostatin-inhibited tumor growth and metastasis in an orthotopic pancreatic cancer model8 and AAV2-mediated snail siRNA inhibited the growth of pancreatic tumor xenograft.9 From these studies, systemic gene therapy appears to be

*Correspondence: Maria Zajac-Kaye, PhD, University of Florida, UF Health Cancer Center, 2033 Mowry Rd, R360, Gainesville, FL 32610. E-mail: [email protected]

HUMAN GENE THERAPY METHODS, VOLUME 28 NUMBER 1 ª 2017 by Mary Ann Liebert, Inc.

DOI: 10.1089/hgtb.2016.089

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effective for both inhibiting tumor growth and preventing metastases in PDAC models.8,9 Moreover, these reports have opened new avenues for the efficient management of pancreatic malignancy using AAV-integrated gene therapy technology.8,9 AAV vectors are recognized as a safe and effective delivery system for gene therapy due to a number of favorable features including lack of pathogenicity, low immunogenicity, lack of viral coding sequences, broad tropism, and the ability to support strong and persistent transgene expression.10,11 Thus far, 12 serotypes of AAV (AAV1 to AAV12) have been studied extensively as gene therapy vectors.12 Various AAV serotype vectors have been successfully applied in over 100 clinical trials with an excellent safety profile.13,14 Among the various serotypes, AAV8 is particularly attractive as a gene therapy vector due to its higher transduction efficiency than other serotype (10- to 100-fold) and lower level of preexisting neutralizing antibodies.15–17 Furthermore, AAV8 crosses vascular endothelial barriers more efficiently than other serotypes, resulting in efficient gene delivery to hepatic, cardiac, and skeletal muscle cells.18,19 The first successful AAV8-mediated gene transfer has been achieved in patients with hemophilia B.20 AAV8 also shows modest transduction of normal exocrine and endocrine pancreas when delivered systemically,21,22 intrapancreatically,23,24 intraductally,22,25 or through the intrapancreatic vessels in animal models.26 The remaining obstacles for pancreatic tropism includes, irrespective of the serotype employed, the persistence of vector dose-dependent immune response and the modest level of transduction efficacy.27,28 A major barrier for AAV gene therapy is the degradation of viral particles during their intracellular trafficking via the ubiquitination-proteasomal degradation machinery.29 To evade phosphorylation and subsequent ubiquitination leading to vector loss, capsid-optimized AAV vectors were developed using site-directed tyrosine to phenylalanine (Y-F) mutagenesis of one or more of the seven surface-exposed tyrosine residues in the viral protein 3 common region of the capsid.30 These Y-F mutant vectors have been reported to protect vector particles from proteasome degradation and yield significant increases in the transduction efficiency of mutant vectors relative to their wild-type (WT) counterparts.30 For example, AAV1, AAV2, AAV3, AAV6, AAV8 or AAV9 containing single or multi-Y-F point mutation sites of the surface-exposed tyrosine residues are significantly more efficient in transducing cells and tissues such as human hematopoietic stem cells, retina, muscle, lung, and liver.30–34 Following these discoveries, capsid mutant AAV vectors have been investi-

gated in proof-of-concept studies for gene therapy in animal models and have been shown to provide longterm retinal preservation,35 correction of murine hemophilia B,36 and restoration of pyruvate dehydrogenase complex activity.37 More recently, the safety and efficacy of capsid-optimized AAV serotype vectors have also been documented in non-human primates38 and human phase 1 clinical trial.14,20,39 In the current study, we used capsid-optimized AAV8 Y-F mutant vectors to promote high transduction efficiency of gene delivery in normal pancreatic tissue and PDAC. We report that a single intraperitoneal administration of a double tyrosine mutant AAV8 (Y447F+Y733F) at a relatively low dose [1–3 · 1011 viral genomes per animal (vg/animal)] exhibits robust transgene expression in normal and malignant mouse pancreas.

MATERIALS AND METHODS Site-directed mutagenesis of AAV8 mutant capsids Tyrosine (Y) to phenylalanine (F) mutant AAV8 capsid were generated by site-directed mutagenesis,40 in a manner similar to previously described generation of AAV2 tyrosine mutants.30 Briefly, Y275F, Y447F, and Y733F mutations were introduced in the pAAV8 capsid plasmid40 using a Multi SiteDirected Mutagenesis Kit (Stratagene, Agilent Technologies, La Jolla, CA) according to the manufacturer’s protocol. Selected tyrosine residues were mutated to phenylalanine to generate the following vectors: a single mutant, Y733F; a double mutant, Y447F+Y733F; and a triple mutant, Y275F+Y447F+Y733F. Phenylalanine was chosen due to similarity in size, opposite charge, and lack of recognition by cellular kinases.29 The presence of the desired point mutation was verified by restriction enzyme analysis and DNA sequencing (Applied Biosystems 3130 Genetic Analyzer; Life Technologies, Warrington, United Kingdom). The amino acids on AAV8 capsid are numbered according to the National Center for Biotechnology Information database (accession ID: NC_006261.1). Generation of recombinant AAV In this study, we used AAV2 genomes pseudotype only in AAV8 capsids, and not in AAV2 capsids; thus the same AAV2 genomes were encapsidated in WT as well as in the mutant AAV8 capsids. A selfcomplementary (sc) AAV8 vector encoding a red fluorescent protein mCherry (sc-smCBA-mCherry) that is driven by the small chicken b-actin (CBA) promoter has been previously described.41 The AAV8 mutant capsids were packaged, purified, and titered

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PANCREATIC TRANSDUCTION BY CAPSID-OPTIMIZED AAV8 VECTORS

according to previously described methods.42 Briefly, recombinant AAV (rAAV) vectors were generated by transient transfection of HEK293T cells using three AAV plasmids (pAAV8-rep/cap-wild type or pAAV8-rep/cap-Y-F mutant, sc-smCBA-mCherry and pHelper).42,43 HEK293T cells were transiently transfected at 80% confluency in forty 150-mm2 dishes using 20 mL of polyethylenimine (linear, MW 25,000, Polysciences, Inc.). Cells were collected 72 hours post-transfection, lysed, and treated with 25 units/mL of benzonase nuclease (Sigma Aldrich, Louis, MO). Subsequently, the recombinant AAV were purified by iodixanol step-gradient ultracentrifugation (Optiprep, Sigma Aldrich), followed by column chromatography (HiTrap Q column, GE Healthcare, Pittsburgh, PA). Recombinant AAV vectors were then concentrated to a final volume of 0.5 ml in phosphate buffered saline using Amicon Ultra 10K centrifugal filters (Millipore, Bedford, MA). The viral DNA-containing AAV vector titers were quantified by real-time PCR analysis and expressed as viral genomes per milliliter (vg/mL). PCR primer sets specific for CBA promoter have been previously described.42,43 A 10-fold dilution series of the control plasmid DNA (sc-smCBA-mCherry) was used to generate standard curve, and AAV signal was compared with the standard curve to determine the AAV vector genome titer. After viral titration, AAV were aliquoted and stored at -80C until used. Animals To generate Pdx1-Cre+, KrasG12D/+, and Ptenlox/+ mice, we backcrossed the KrasG12D/+ line (on a B6.129 background, NCI Mouse Repository No. 01XJ6) to the Ptenlox/+ mice (on a 129/BALB/c background, Jackson lab No. 004597). We then crossed KrasG12D/+;Ptenlox/+ mice to Pdx1-Cre+ mice (on a B6/FVB background, NCI Mouse Repository No. 01XL5).44 All experimental mice (FVB/C129; Pdx1-Cre+, KrasG12D/+, and Ptenlox/+) were housed and handled at the University of Florida Animal Facility. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Florida. Recombinant AAV8 transduction studies in vivo WT and Y-F mutant AAV8 (WT-AAV8 and Y447F+Y733F-AAV8) were administered via the tail vein or intraperitoneal (i.p.) injection into 2-month-old FVB/C129 mice at a dose of 1 · 1011 vg/ animal. Mice were euthanized 2 weeks after AAV administration. Mouse tissues were assessed for mCherry expression either by ex vivo imaging or by fluorescence microscopy as described in sections

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below. Additionally, WT and Y447F+Y733F-AAV8 were intraperitoneally administered at a dose of 3 · 1011 vg/animal into 2-month-old KrasG12D/+/ Pten+/- or 5-month-old KrasG12D/+ mice. Two weeks after AAV injection, examination of mCherry expression and viral genome copy number on normal or tumor tissues was performed following euthanasia (see below). Examination of mCherry expression Ex vivo visualization of mCherry was performed in different organs as follows. Internal organs were collected, placed on black paper (Artagain black paper, Strathmore cat No. 445-109), and the fluorescence intensity was measured using IVIS Imaging System (Caliper Life Sciences, Hopkinton, MA). In vivo imaging system (IVIS) data are presented as radiant efficiency [(p/s/cm2/sr)/(lW/cm2)]. Radiant efficiency is the number of photons (p) per second (s) that leave a square centimeter (cm2) of tissue and radiate into a solid angle of one steradian (sr) normalized to the incident excitation intensity (lW) per square centimeter. Quantification of fluorescent signals from organs was analyzed by Living-Image 4 software (Caliper Life Sciences). Cross-sections from liver and pancreas were used to assess mCherry expression by a fluorescence microscope (Leica CTR6000; Leica Microsystems GmbH, Stuttgart, Germany). Briefly, individual organs were collected, fixed in 4% paraformaldehyde for 24 hours, immersed in 30% sucrose for 24 hours, mounted in optimal cutting temperature (OCT) medium (Tissue-Tek, Torrance, CA), and snap frozen. Cryosections were cut (Leica, Cryostat CM30505) and mounted in DAPI-containing fluorescence mounting media (Invitrogen Molecular Probes, Eugene OR). Fluorescent images of cryosection were recorded using an Olympus DP70 digital camera coupled to an Olympus IX71 inverted microscope (Olympus Corp, Japan). Fluorescence expression efficiency was measured using Image J software. Real-time PCR for AAV genome copy number determination At necropsy, the pancreas and liver were frozen in OCT medium and stored at -80C until genomic DNA was extracted. Total DNA was extracted with a DNAeasy Blood and Tissue kit (Qiagen, Valencia, CA). Vector genome copy number was determined with a 7900 HT real-time PCR system (Applied Biosystems, Foster City, CA). TaqMan assays for viral vector genome copy number were developed using primers and probe for the CBA promoter region. Forward primer is TCTGCTTCACTCTCCC

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CATCTC. Reverse primer is CCATCGCTGCACA AAATAATTAAA. Fluorescent probe is 6FAM (6carboxyfluorescein)-CCCCCTCCCCACCCCCAATT. sc-smCBA-mCherry plasmid42,43 was used to produce a standard curve. PCR reactions contained a total volume of 100 lL and were run under the following conditions: 50C for 2 minutes, 95C for 10 minutes, 45 cycles of 95C for 15 seconds, and 60C for one minute. DNA samples were assayed in triplicate. In order to rule out false negatives due to PCR inhibition by viral DNA, the third replicate was ‘‘spiked’’ with positive control plasmid DNA (sc-smCBA-mCherry) at a concentration of 100 copies/g of total genomic DNA. The AAV vector genome copy number is normalized as viral genomes copy number per lg of total genomic DNA. AAV vector genome copy number determination procedures were performed at the Powell Gene Therapy Center Vector Core facility at University of Florida. Statistical analysis A two-tailed Student’s t-test determined probability of difference. A p value